Nucleic acid amplification techniques such as polymerase chain reaction (the reaction may be abbreviated to PCR hereinafter) are innovative in that the techniques are able to amplify a target DNA region 100,000 times or more in a short time. However, it is difficult to optimize the reaction. There is a technical issue that the amplification specificity is reduced by non-specific amplification caused by mis-annealing of the primer such as annealing the primer to sites other than the target sequence or annealing between primers. The amplification product which is not specific to a template nucleic acid could be a factor to reduce the amplification specificity and cause background noise to affect experiments to be conducted later. Therefore, it is required to establish an amplification technique having high accuracy for restraining the non-specific amplification and specifically amplifying a target nucleic acid.
A variety of attempts are reported for controlling the reaction to prevent the mis-annealing of the primer at each step of the amplification cycle. Specifically, it has been reported that the nucleic acid amplification specificity is improved by carrying out PCR in the presence of single strand binding proteins (referred as to SSB hereinafter) derived from Thermus thermophilus, SSB derived from E. coli, or SSB from bacteriophage T4 (refer as to T4gp32 hereinafter) (T4 gene 32 protein, refer to Non patent documents 1, 2, 3). It is also reported that the nucleic acid amplification specificity is improved by carrying out PCR by a DNA polymerase to which a double-stranded DNA binding protein (Sso7d) is bound (for example, refer to Patent document 1 and Non patent document 4). However, these methods do not fully satisfy the needs of the market in terms of the amplification specificity.
Recently, the inventors reported that a RecA protein derived from an extremely thermophilic bacterium can bind to a template or a primer to promote binding of the primer only to a specific template sequence and the mis-annealing of the primer can be restrained thereby (For example, refer to Patent documents 2 and 3, and Non patent document 5). A RecA protein binds to a single-stranded nucleic acid cooperatively, searches a homologous region between the single-stranded nucleic acid and a double-stranded nucleic acid and undergoes homologous recombination of nucleic acids.
However, in the above-method that the inventors reported, it was found that biological functions of the heat-stable RecA protein were not fully achieved depending on the reaction condition and the non-specific amplification might occur. Thus, in order to achieve the high specificity of nucleic acid amplification, an improved technique is still needed for properly controlling the nucleic acid amplification reaction. Also, the nucleic acid amplification reaction requires expensive reagents such as a thermostable DNA polymerase. For this reason, a technique is needed for reducing the amount of the expensive reagents and specifically amplifying the target nucleic acid in an inexpensive manner.
A need exists for a heat-stable RecA mutant protein and a nucleic acid amplification method using the heat-stable RecA mutant protein which are not susceptible to the drawback mentioned above.
Patent Document 1: WO 04/037979
Patent Document 2: U.S. Pat. No. 2005/260631
Patent Document 3: EPO Publication 1522597
Non-Patent Document 1: Perales et al., “Enhancement of DNA, cDNA synthesis and fidelity at high temperatures by a dimeric single-stranded DNA-binding protein.” Nucleic Acids Research, Volume 31, 22th, 6473-6480, 2003
Non-Patent Document 2: Chou Q., “Minimizing deletion mutagenesis artifact during Taq DNA polymerase PCR by E. coli SSB.” Nucleic Acids Research, Volume 20, 16th issue, 4371, 1992
Non-Patent Document 3: Rapley R., “Enhancing PCR amplification and sequencing using DNA-binding proteins.” Mol. Biotechnol., volume 2, 3rd issue, 295-298, 1994
Non-Patent Document 4: “iProof High-Fidelity DNA polymerase” [online], BioRad Laboratories Inc., [Jan. 6, 2006 searched] internet <URL: HYPERLINK
Non-Patent Document 5: Shigemori Y. et al., “Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products.” Nucleic Acids Research, Volume 33, 14th issue, 2005, e126